Novel coronavirus associated with severe respiratory disease - … Further information...

Novel coronavirus associated

with severe respiratory

disease - Laboratory aspects

Dr. Janice Lo

Public Health Laboratory Services Branch

Centre for Health Protection

Department of Health

Background

20 September 2012: Isolation of novel

coronavirus from patient in Saudi Arabia

M/60 years: Pneumonia associated with

acute renal failure

Virus grows readily on Vero and LLC-MK2

cells: Rounding and syncytia formation

Pan-coronavirus RT-PCR: Positive

Netherlands laboratory: Confirmed new

member of beta group of corononavirus,

closely related to bat coronaviruses

Further information

Eurosurveillance 27 September 2012

Saudi Arabia case reported in Promed:

13 June 2012: Presented with pneumonia

24 June 2012: Passed away

Qatar case:

M/40+

11 September 2012: Evacuated to United Kingdom

for management of pulmonary and renal failure

Pan-coronavirus PCR positive for coronavirus;

nucleotide sequence closely matched with virus

from above case

Cases 3-6

Case 3:

Saudi Arabia

Recovered

Case 4:

Qatar

Not fatal

Case 5 and 6:

Saudi Arabia

Epidemiologically linked (family members)

One fatal, one recovered

Cases 7-9

Case 7:

Saudi Arabia

Occurred in October 2012

Epidemiologically linked to cases 5 and 6

Fatal

Case 8-9:

Jordan

April 2012

Epidemiologically linked to each other

Both fatal

European CDC Communicable

Disease Threats Report

Cases 8 and 9

April 2012: A number of severe pnemonia

cases in Jordan

WHO Collaborating Centre for Emerging

and Re-emerging Infectious Diseases

(NAMRU – 3) provided testing: All

samples negative for known

coronaviruses/other respiratory viruses

October 2012: Stored samples sent by

Jordan to NAMRU-3, confirmed two novel

coronavirus infection cases in November

Summary

First known case infected in April 2012

(Jordan)

So far confined to Middle East

Jordan: 2/2 fatal

Qatar: 0/2 fatal

Saudi Arabia: 3/5 fatal

Evidence of limited person-to-person

transmission

Virus 2009; 1: 57

Virus 2009; 1: 57

Diagnostic strategy – Initial

Pan-coronavirus reverse-transcription

polymerase chain reaction

Nucleotide sequencing

Sequence analysis

Turnaround time:

Negative – 1 day

Positive – 2 days

Diagnostic strategy – Subsequent

Real-time reverse-transcription

polymerase chain reaction

UpE

Orf1b

Considerations

Positive control

Specimen type

Timing of specimen

13 November 2012

Eurosurveillance 6 December 2012

Diagnostic tests

Real-time reverse-transcription PCR

UpE

Orf1b

Orf1a

Sequencing targets (conventional hemi-

nested PCR)

RdRp (Orf1b) – Not for screening; primers

cross-react with other coronaviruses

N (6 nt deletion in London strain) – Specific

for novel virus; possibly for strain

classification

Other tests

Virus isolation

Technically feasible

Biosafety considerations

Positive isolate as early as 2 days after

inoculation

Confirmation using PCR

Other tests

Antibody detection

Biosafety considerations

Immunofluorescence (Western blot,

neutralization)

Spike and nucleocapsid antibodies

For cases with no respiratory specimens within

the first 10 days after onset

Paired acute and convalescent titres

Possibility of cross-reactivity

Indications for testing

Clinical suspicion: Severe respiratory

disease; renal failure

Epidemiological consideration: Travel

history; contact history?

Obtain specimens based on disease

course

Liaise with laboratory as necessary

Thank you